Small extracellular vesicles from irradiated lung epithelial cells promote the activation of fibroblasts in pulmonary fibrosis

AbstractBackground Alveolar epithelial injury and dysfunction are the risk factors for radiation-induced pulmonary fibrosis (RIPF). However, it is not clear about the relationship between RIPF and the small extracellular vesicles (sEV) secreted by irradiated alveolar epithelial cells. Based on the activation of fibroblasts, this study explored the role of sEV derived from alveolar epithelial cells in RIPF and the potential mechanisms.Methods Transmission electron microscopy (TEM), nanoparticle tracking analysis (NTA), and western blotting were used to characterize sEV. Western blotting was used to detect fibrosis-associated proteins. Cell counts and transwell assays were used to evaluate the proliferation and migration ability of fibroblasts. RT-PCR was used to observe the extracellular matrix (ECM) synthesized by fibroblasts, miRNA changes in the sEV were determined by second-generation sequencing.Results TEM, NTA, and western blotting showed the extracellular vesicles with a double-layer membrane structure of approximately 100 nm in diameter. The sEV derived from irradiated A549, HBEC3-KT, and MLE12 cells upregulated FN1 and alpha-SMA proteins expression in fibroblasts and drove the fibroblast to myofibroblast transition, and the sEV from irradiated mouse bronchoalveolar lavage fluid (BALF) affirmed the same results. In addition, the sEV derived from irradiated alveolar epithelial cells significantly increased the migration ability of fibroblasts and the expression of extracellular matrix proteins such as FN1. The results of miRNA sequencing of sEV in BALF of rats with RIPF showed that the metabolic pathway may be important for miRNA to regulate the activation of fibroblasts.Conclusion The sEV derived from radiated pulmonary epithelial cells promote the activation, migration and extracellular matrix proteins expression of lung fibroblasts; miRNA in sEV may be an important molecular that affects the activation of lung fibroblasts.Keywords: Radiation-induced pulmonary fibrosissmall extracellular vesicleslung epithelial cells Authors contributionsConceptualization, Liqing Du; Data curation, Na Li, Kejun Li, and Wenyue Zhao; Formal analysis, Na Li and Kejun Li; Funding acquisition, Qiang Liu; Methodology, Na Li, Kejun Li and Wenyue Zhao; Software, Kaihua Ji, Ningning He, Jinhan Wang, and Manman Zhang; Supervision, Liqing Du and Qiang Liu; Validation, Yan Wang, Chang Xu, Qin Wang, and Yang Liu; Visualization, Na Li and Kejun Li; Writing – original draft, Na Li and Kejun Li; Writing – review & editing, Liqing Du.Disclosure statementNo potential conflict of interest was reported by the author(s). This study was independently conducted by the author in accordance with the author's contributions, without any conflict of interest.Additional informationFundingThis study was supported by the National Natural Science Foundation of China (31971168, 81972976, 31900891, 32071241, 82072331, 82103837, 32171239, 31900889, 82273580, 32201015), the Fundamental Research Funds for the Central Universities (3332021066), Natural Science Foundation of Tianjin (19JCYBJC26600 and 21JCYBJC01510), CIRP Open Fund of Radiation Protection Laboratories (CIRP-RRPE20220201 and CIRP-DTRI20220202).Notes on contributorsNa LiNa Li, Faculty of the Department of Radiation Biology student, Institute of Radiation Medicine, Chinese Academy of Medical Science and Peking Union Medical College, Tianjin, China.Kejun LiKejun Li, Faculty of the Department of Radiation Biology student, Institute of Radiation Medicine, Chinese Academy of Medical Science and Peking Union Medical College, Tianjin, China.Wenyue ZhaoWenyue Zhao, Faculty of the Department of Radiation Biology student, Institute of Radiation Medicine, Chinese Academy of Medical Science and Peking Union Medical College, Tianjin, China.Yan WangYan Wang, PhD, Professor, Department of Radiation Biology, Institute of Radiation Medicine, Chinese Academy of Medical Science and Peking Union Medical College, Tianjin, China.Chang XuChang Xu, PhD, Professor, Department of Radiation Biology, Institute of Radiation Medicine, Chinese Academy of Medical Science and Peking Union Medical College, Tianjin, China.Qin WangQin Wang, PhD, Professor, Department of Radiation Biology, Institute of Radiation Medicine, Chinese Academy of Medical Science and Peking Union Medical College, Tianjin, China.Lifeng PanLifeng Pan, MD, Chief physician, Department of general surgery, Chu Hsien-I Memorial Hospital of Tianjin Medical University, Tianjin, China.Qiang LiQiang Li, M.M, Attending doctor, Department of general surgery, Chu Hsien-I Memorial Hospital of Tianjin Medical University, Tianjin, China.Kaihua JiKaihua Ji, PhD, Associate Professor, Department of Radiation Biology, Institute of Radiation Medicine, Chinese Academy of Medical Science and Peking Union Medical College, Tianjin, China.Ningning HeNingning He, PhD, Associate Professor, Department of Radiation Biology, Institute of Radiation Medicine, Chinese Academy of Medical Science and Peking Union Medical College, Tianjin, China.Yang LiuYang Liu, PhD, Associate Professor, Department of Radiation Biology, Institute of Radiation Medicine, Chinese Academy of Medical Science and Peking Union Medical College, Tianjin, China.Jinhan WangJinhan Wang, PhD, Associate Professor, Department of Radiation Biology, Institute of Radiation Medicine, Chinese Academy of Medical Science and Peking Union Medical College, Tianjin, China.Manman ZhangManman Zhang, PhD, Assistant Researcher, Department of Radiation Biology, Institute of Radiation Medicine, Chinese Academy of Medical Science and Peking Union Medical College, Tianjin, China.Mengmeng YangMengmeng Yang, MD, Senior Technician, Department of Radiation Biology, Institute of Radiation Medicine, Chinese Academy of Medical Science and Peking Union Medical College, Tianjin, China.Liqing DuLiqing Du, PhD, Professor, Department of Radiation Biology, Institute of Radiation Medicine, Chinese Academy of Medical Science and Peking Union Medical College, Tianjin, China.Qiang LiuQiang Liu, PhD, Professor, Department of Radiation Biology, Institute of Radiation Medicine, Chinese Academy of Medical Science and Peking Union Medical College, Tianjin, China.